Developments in molecular biology have provided researchers with tools for constructing recombinant DNA in vitro. Several applications of this technique include: (1) investigating chromosome structure; (2) analyzing mechanisms of genetic regulation, (3) isolating specific genes or segments of DNA, and (4) constructing intergeneric chimeras with unique properties and biosynthetic capabilities. Recent reports of the production of insulin and somatostatin using techniques of recombinant DNA are examples of the significant impact of recombinant DNA research.
To assist in understanding the new terminology of this field, the following definitions of terms used throughout this application are provided:
DNA--Deoxyribonucleic acid; PA0 Bacteriophage--any of the viruses that infect bacterial cells; also known as phage; a particle composed of a piece of DNA enclosed and contained within a protein head portion and having a tail and tail fibers composed of protein; PA0 Transducing phage--A bacteriophage that carries fragments of bacterial chromosomal DNA and transfers this DNA on subsequent infection of another bacterium; PA0 Transduction--Bacteriophage-mediated transfer of bacterial genetic information; PA0 Donor--In transduction, the strain used for propogation of phage to be used in transducing markers to a recipient; PA0 Recipient--In transduction, the strain which receives genetic markers by transduction; PA0 Chimera--A strain carrying DNA which contains genetic information from two different sources; PA0 Transductant--A new strain generated by transduction; PA0 Auxotroph Mutant--A mutant which requires one or more growth factors in the medium not required by the parent cells; PA0 Phototroph--An organism which does not require a growth factor in the media; PA0 Phenotype--The observable character of an organism controlled by the gene; PA0 Genome--The total genetic endowment of a species; PA0 pfu--Plaque-forming units, i.e., the number of infective bacteriophage; PA0 moe--Multiplicity of exposure; i.e., the number of pfu divided by the number of cells; PA0 NIH Guidelines--The National Institutes of Health safety recommendations for research involving recombinant DNA molecules; PA0 P1, P2, P3, and P4--Physical-containment levels established by the NIH Guidelines; P1 being the lowest level of containment and P4 being the most stringent level of containment; PA0 EK1, EK2, and EK3--Biological-containment levels established by the NIH Guidelines; EK1 being the lowest level of containment and EK3 being the most stringent level of containment.
Experiments with recombinant DNA are regulated by guidelines that have been established by the NIH. Many important experiments require effective containment to reduce the probability that experimental chimeras will escape from the laboratory. Under the current NIH guidelines biological containment is to be used in combination with physical containment for safety purposes. Biological containment requires the use of a crippled strain of Escherichia coli, such as E. coli K12. E. coli K12 is a laboratory-adapted strain unable to survive more than 48 hours in the human intestinal tract. Several planned mutational defects in E. coli K12 have resulted in a self-destructing E. coli strain called E. coli K12 .chi.1776. This strain is acceptable for use in experiments requiring an EK2 host-vector system. E. coli K12 .chi.1776 is unable to synthesize its own cell wall or to replicate its DNA outside a carefully controlled laboratory environment. In addition to the fact that its ability to survive in the human intestinal tract is further reduced, E. coli K12 .chi.1776 is also extremely sensitive to (1) sunlight, (2) moderately warm temperatures and (3) detergents and chemicals which are frequently found in sewers and polluted waters. For this reason, E. coli K12 .chi.1776 (herein called .chi.1776) has been approved as an EK2 host for biological containment.
In the combination of physical and biological containment outlined in the NIH guidelines, an EK2 host can be used in experiments at a reduced level of physical containment compared with similar experiments using an EK1 host.
The normal experimental protocol for transducing E. coli strains such as E. coli K12 involves accomplishing transduction during the exponential growth phase. Because of the importance of E. coli K12 .chi.1776, the ability to transduce genetic markers into this strain is extremely important. Derivatives of .chi.1776 for this purpose, however, could not be isolated using the procedure for transducing E. coli K12. Heretofore, transduction of E. coli during the stationary growth phase was unknown. I have discovered that in cultures of E. coli, such as .chi.1776, that are transduced poorly in the exponential growth phase, transduction can be accomplished in the stationary growth phase.